The study of RNA folding reactions has paralleled, and benefited significantly from, the DNA studies being conducted as Core research in projects 1 &2. The Mg2+ bound and Mg2+ free forms of the RNA ribozyme exhibit distinctly different patterns of hydroxyl radical reactivity. Thermodynamic analysis of ribozyme folding. The Tetrahymena ribozyme adopts a compact tertiary structure upon the addition of divalent cations. Protection from OH cleavage is believed to predominantly result from the decreased solvent accessibility of the phosphodiester backbone within the interior of the folded ribozyme. Thus, the isotherms and progress curves determined from the OH protection data are interpreted to reflect the folding of the ribozyme. It is assumed in this analysis that the rate of OH cleavage of RNA is linearly related to the solvent accessibility of the phosphodiester backbone. Quantification of individual sites of protection as a function of [Mg2+] reveals that the domains of the ribozyme bind Mg2+ with unique values for cooperativity and/or stoichiometry. The three sites analyzed within the P3-P7 domain are all characterized by nH 3 4, consistent with the high Mg2+ cooperativity observed in other studies.The equivalence of nH values for the three sites within the P3-P7 domain is indicative of the highly concerted nature of Mg2+ binding by the ribozyme. The high cooperativity of the Mg2+-dependent folding of the P3-P7 domain contrasts with the more moderate values (nH values clustering around 2) determined for the other domains of the ribozyme; these values are comparable with the "average" value of nH & 3 determined previously. The conclusion that can be drawn from these results is that the effects of Mg2+ ion binding are not uniformly distributed throughout the ribozyme. It is tempting to speculate that the binding of an additional Mg2+ ion is linked to the folding of the P3-P7 domain since nH cannot exceed the stoichiometry of the binding reaction. (However, identical Mg2+ stoichiometries with the cooperativity of Mg2+-dependent folding being more highly concerted in the P3-P7 domain is an interpretation not excluded by the data.) The suggestion that an additional Mg2+ ion is linked to the folding of the P3-P7 domain is consistent with the conclusion that Mg2+ binding follows the rate-limiting folding of P3 to form the active ribozyme. It is tempting to speculate that the additional Mg2+ ion(s) stabilize the ribozyme core. Phosphorothioate substitution and rescue by Mn2+ has identified several divalent cation binding sites on the ribozyme, for example, along the phosphodiester backbone of the J8/7 single stranded region. In the three-dimensional model of the Tetrahymena ribozyme core) this highly conserved region is buried deep in the core of the domain and forms the interface between the P4-P6, P3-P7 and P1 domains. Kinetic analysis of ribozyme folding. The results developed in the last year demonstrate that time-resolved synchrotron x-ray "footprinting" is sensitive to time-dependent changes in local regions of OH protection of RNA. The rate of formation of tertiary interactions is followed in this assay by measuring changes in the accessibility of local regions of the RNA to the OH probe. The internal consistency of the OH kinetics data, as will be shown below, and the consistency of the OH kinetics and hybridization-competition studies provides compelling evidence that these approaches accurately monitor the RNA folding reaction. We will continue these studies at sub second timescales to better understand the folding process.